Vascular endothelium responds to shear stress due to blood flow by releasing nitric oxide (NO). Deficiencies in this vasodilator has been implicated in diseases such as essential hypertension, coronary artery insufficiency and chronic heart failure. However, Because of the short T1/2 of this free radical (<6 Sec.) the mechanism by which shear stress induces NO release and the components of the shear stress activated pathway are unknown. We propose to investigate this mechanism using our unique published porphyrin/Nafion coated carbon fiber microsensor for on line detection of NO combined with fura-2 fluorescence and patchclamp techniques for monitoring Ca2+ flux. Shear stress elevates cytosolic Ca2+ ([Ca2+]i) which is necessary for the activation of Ca2+<Calmodulin sensitive endothelial NO synthase (cNOS). However, it is unclear whether the source(s) of this Ca2+ is influx across the plasmalemma (mechano-sensitive non-selective cation channels) and/or release from intracellular stores (mechano-sensitive detectors or extravasated ATP activating surface P2 purinoceptors). Furthermore, it has been reported that chronic shear stress up-regulates cNOS and that evolved NO modulates it own release. We propose to test the hypothesis that shear stress induces NO release in a Ca2+ dependent manner via a pathway that is up-regulated by its initiating signal (shear stress) and down-regulated by its end product (NO). Experiments will be conducted on intact or permeabilized bovine aortic endothelial cells (BAECs), employing concurrent measurements of single channel or whole-cell transmembrane currents and [Ca2+]i or NO fluxes during varying levels of shear stress or in the presence of compounds (P2 purinoceptor agonist ACP or Ca2+ ionophore A23187) known to elicit NO release.